Fish increasingly attract public, scientific and political interest around the world. Recreational and commercial fisheries are prominent in our societies; fish issues take a highly relevant position in discussions related to conservation biology (1) and environmental protection efforts (e.g., effects of climate change, novel predators, novel animal-environment relationships on stress and as a consequence on fitness) (2, 3). Anthropogenic activities (e.g., energy production, shipping traffic, industrial pollution) compromise wild stocks (4). Therefore, various international monitoring schemes aim to scientifically clarify their impact on the health status of oceanic niches. A similar situation exists for freshwater stocks as they are under threat by soil erosion, fertilizers, etc., as agriculture expands. The human population keeps on expanding, making the need for a sustainable food production a prime global priority. Fish protein is one of the most important protein sources for human consumption. Now that fisheries meet limits in yield, aquaculture expands rapidly worldwide and puts increasing pressure on farmers to produce in an optimal, sustainable and animal-friendly way (5, 6). High numbers of fish (e.g., zebrafish and medaka) are used as vertebrate models and alternatives to rodents in biomedical research (e.g., in bone physiological research). The imperative to maintain research-driven innovation in this sector, while global economic output dwindles, adds to exploitation of fish. In the recent past, ethics concerning fish suffering and welfare urge more attention as high numbers of fish are involved in fisheries (7), the rapidly growing and intensifying aquaculture industries (8, 9), public aquaria (10, 11) and scientific research laboratories (12). Appreciating and understanding fish biology as basis for management, control and decision-making in fish exploitation puts a phenomenal challenge to those involved, coming from a multitude of disciplines (from molecular biology to eco-physiology), viewing from multiple angles (all different stakeholders) and representing highly specific and cherished expertise. In this framework, new scientifically validated biomarkers to assess levels of stress in fish, in particular, of chronic stress, are of utmost importance.
Fish welfare easily becomes compromised, and consensus is growing that proper welfare assessment requires animal-based, physiological indicators being superior to less consistent, indirect husbandry-related parameters such as water quality. However, a shortage exists on practical, reliable and validated biomarkers for chronic stress. A frequently used, seemingly logical biomarker for fish is the blood level of the “stress steroid” cortisol. Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol (13, 14) into the blood. Cortisol elicits a suite of physiological and behavioral changes (15-17) that allow the fish to cope with altered situations (18-20). The adaptive value of short-term cortisol actions is widely recognized (21, 22). Far less is known about persistent stress and its mostly detrimental consequences for health, growth, and reproduction (19, 20). Definition of a robust, easily performable and scientifically validated chronic stress biomarker is thus of utmost importance. Glucocorticoid levels in plasma of fish show diet variation (23), do not reveal the lifetime exposure of the fish to stress, and provide no more than a snapshot of the cortisol status at the moment of sampling (24, 25). Moreover, blood sampling is invasive and unavoidably causes confounding stress to the fish because of netting, air exposure and handling. This makes plasma cortisol prone to bias as levels rise rapidly a few minutes after confrontation with a stressor (13). Anesthetics adopted to facilitate blood sampling may, by themselves, reduce or block the activation of the HPI-axis, thereby affecting the cortisol release in blood and resulting in erroneous results (26, 27). Restrictions also apply to the assay of cortisol in alternative matrices such as mucus (28, 29), gut content (29), feces (30) and water (31, 32). The pertinent literature lacks data on cortisol in a matrix suitable for chronic stress evaluation. Hitherto, the majority of studies addressed cortisol only, and reports on glucocorticoid production pathway(s) (33) or their significance in chronic stress are scarce. Fish record (a-)biotic events and store this history in calcified tissues (34-37). As feathers of birds (38) and hair of mammals (39), the ideal matrix for chronic stress assessment in fish should at least meet the following criteria: (i) incorporation of glucocorticoids; (ii) slow but persistent growth; and (iii) ease in sampling.
Elasmoid scales, calcified dermal exoskeletal structures (40), grow along with the fish and consist of an acellular collagenous matrix, which is mineralized with calcium hydroxyapatite on the outer layer and lined with a monolayer of cells with osteoblast- and osteoclast-like properties. Upon removal, a scale will regenerate within days (41). Scales are a target for endocrine stimuli. A high affinity, low-capacity estradiol-17b binding was found in scleroblast cytosol of rainbow trout (Oncorhynchus mykiss) (42) and estrogen receptors have immunohistochemically been detected in Mozambique tilapia (Oreochromis mossambicus) and gilthead sea bream (Sparus auratus) scales (43). A scale is easily and quickly collected with negligible injury and stress to the fish without confounding its cortisol levels due to the sampling procedure.